Dna gridiron compositions and methods

ABSTRACT

Novel compositions and methods for engineering wireframe architectures and scaffolds of increasing complexity by creating gridiron-like DNA structures (FIG. 1). A series of four-arm junctions are used as vertices within a network of double-helical DNA fragments. Deliberate distortion of the junctions from their most relaxed conformations ensures that a scaffold strand can traverse through individual vertices in multiple directions. DNA gridirons, ranging from two-dimensional arrays with reconfigurability to multilayer and three-dimensional structures and curved objects, can be assembled according the methods presented herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/202,841, filed on Nov. 28, 2018, which is a continuation of U.S.patent application Ser. No. 15/121,007, filed on Aug. 23, 2016, which isa national stage entry of International Patent Application No.PCT/US2015/017553, filed on Feb. 25, 2015, which claims priority to U.S.Provisional Patent Application No. 61/944,677 filed on Feb. 26, 2014.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under N000140911118awarded by the Office of Naval Research, 1104373 awarded by the NationalScience Foundation, and W911NF-11-1-0137 awarded by the Army ResearchOffice. The government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Sep. 25, 2020, is namedG8118-00304_sequence_listing.txt and is 53,172 bytes in size.

FIELD OF THE INVENTION

This disclosure relates to the field of nanotechnology and moreparticularly to the engineering of wireframe architectures and scaffoldsusing DNA structures.

BACKGROUND OF THE INVENTION

Self-assembling nucleic acid molecules have shown merit as versatilematerials for organizing and constructing complex nano-scale structures.Methods are known for generation of complex DNA origami nanostructureswith addressable surface features. For example, a long scaffold strand,most often the 7429-nucleotide (nt) circular genome of the M13mp18bacteriophage, is organized and folded by interactions with a largenumber of short, synthetic, staple strands. The path of the scaffoldstrand in this approach has been restricted to discrete domains ofparallel lines because it is based on the double crossover unit motif tolink adjacent helices.

Because engineering wireframe architectures and scaffolds of increasingcomplexity is an important challenge in nanotechology, methods andcompositions for achieving same are very useful and inventive.

SUMMARY OF THE INVENTION

We present a design strategy that uses an unusual set of immobileHolliday junction analogs (four-arm junctions) as the basic structuralunit of DNA origami nanostructures and as joints to construct a varietyof two-dimensional (2D) and 3D gridiron structures, in which thescaffold strand and corresponding double helices are not restricted to a1D parallel, raster-fill pattern. By programming the connection betweenindividual joints with DNA segments of variable lengths, we constructedcomplex wireframe geometries.

These and other aspects of the invention will be apparent upon referenceto the following detailed description and figures. To that end, anypatent and other documents cited herein are hereby incorporated byreference in their entirety.

BRIEF DESCRIPTION OF THE FIGURES

The patent application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1. (A) (Left) Geometry and strand polarity of a single gridironunit formed from four four-arm junctions. (Right) Geometry and polarityof a double-crossover molecule motif used in conventional DNA origamistructures. For both structures, the ssDNAs depicted in red arecomponents of DNA double helices that serve as the scaffold strands. ThessDNA depicted in gray represents staple strands. (B) Models of fourfour-arm junction molecules in their relaxed conformation. Theorientation of the upper two junctions differs from that of the lowertwo by a 180° in-plane rotation. Thus, the polarities of the continuousred strands in the upper and lower layers of the horizontally orientedhelices are antiparallel to one another. (C) Models illustrating thedeviation from a relaxed conformation required of the four individualjunctions to form a gridiron unit. The blue arrows indicate that the tophelix of the junctions in the upper-left and lower-right corners must berotated 150° clockwise, whereas in the upper-right and lower-leftjunctions they must be rotated 30° counterclockwise. (D and E) Helicalmodels illustrating a complete gridiron unit. (F and G) Schematicsillustrating a typical scaffold-folding path for a 2D DNA gridironpattern.

FIG. 2. (A to D) Images for a 2D gridiron structures with 21-bp cavitieswith AFM [(A) and (B)] and TEM images [(C) and (D)]. (E and F) Imagesfor a 2D gridiron with 63-bp cavities with AFM (E) and TEM images (F).(G to J) Schematics (left), TEM images (middle), and histogram analysis(right) of the angle distributions for angle control. All scale barsindicate 200 nm, and all zoom-in images (images without scale bars) are200 by 200 nm.

FIG. 3. Multilayer gridiron design strategies. (A and B) Strategy 1 isstacked layers. (A) A portion of a double-layer gridiron lattice with52-bp cavity size. The yellow circles designate the permissibleconnection points to a third layer. The dashed lines correspond topossible connection points to form additional layers. (B) Given thedouble-layer gridiron lattice (X and Y lengths) and the distance betweencrossover points in the third layer, the angle q can be calculated as180°−cos−1[(X2+Y2−L2)/2XY]. (C) Strategy 2 is intertwining gridironplanes. (D to F) Schematics (left), AFM (middle), and TEM (right) imagesof (D) a three-layer hexag-onal gridiron design, q=120°; (E) afour-layer gridiron design, q is not controlled because the dashed greenline in (A) represents a connection strategy that cannot fix the angle;and (F) a 3D gridiron assembled by using strategy 2. All scale barsindicate 200 nm, and all zoom-in images (images without scale bars) are200 by 200 nm.

FIG. 4. Schematics (left), AFM (middle), and TEM images (right) of (A)an S-shaped structure, (B) a sphere, and (C) a screw. All scale barsindicate 200 nm, and all zoom-in images (images without scale bars) are200 by 200 nm. In (B) and (C), the diameter and the width, respectively,appear to be larger in the AFM images compared with the TEM images. Thisdifference is probably a result of flattening of the 3D objects intotwo-layer structures and AFM tip convolution.

FIG. 5 shows sequences of the staples in the 21 bps Gridiron structure.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Although intuitively one could imagine threading a single-strandedscaffold through a number of four-arm junction units in both horizontaland vertical directions to create gridiron like patterns, the structuralproperties of traditional Holliday junction impose certain challengesthat require unconventional arrangement of the junction unitconformation, as revealed by the design principles described below.

We compared a gridiron unit to a double crossover motif (FIG. 1A), andthe DNA strands am abstracted to display only their polarity with thearrows pointing from 5′ to 3′. In the gridiron unit, four four-armjunctions are linked together to form a two-layer square frame in whichthe helices on opposite sides lie in the same plane. An antiparallelarrangement between opposite sides of the square frame permits a single,central strand to traverse each of the helices.

Each of the four junctions is depicted in its relaxed conformation (FIG.1B) such that the helices form a right-handed twist with a 60° torsionangle. Deviation from a relaxed conformation is required of eachjunction to form the gridiron unit cell. First, the red strands in thehorizontally oriented helices (both top and bottom images) can be linkedtogether to produce continuous strands without reversing the 5′-to-3′polarity (FIGS. 1, B and C). Next, the vertically oriented helices needto be rotated in the plane about the junction points (FIG. 1C) to allowthe formation of continuous 5′-to-3′ connections between upper and lowerjunctions (FIGS. 1, D and E).

Connecting a number of gridiron units leads to the formation of avariety of 2D lattices (FIGS. 1, F and G). The red lines represent theDNA strands that are expected to retain an unperturbed helical structurewith continuous base stacking. Meanwhile, the short strands (in gray)form the crossovers between helical domains and function as staples. Along scaffold strand is created by connecting the termini of the redstrands with short single-stranded DNA (ssDNA) loops. In the most basicdesign, the scaffold begins at one corner, fills the first layer,changes direction at the opposite corner, and then fills the secondlayer to produce a structure in which the helices within the two layersare oriented perpendicularly with respect to each other. Lastly, thescaffold returns to its initial position to form a closed loop (FIG.1G).

The cavity size of gridiron structures can be tailored by altering thenumber of base pairs between the adjacent junction points. An 11-by-11gridiron structure (11 vertical helices by 11 horizontal helices) with21 base pairs (bp) between junctions in both directions uses 5301 of7249 nt of the M13mp18 ssDNA scaffold strand and contains 120 staplestrands (42 nt each). The remaining 1948 nt of the scaffold form asingle-stranded loop at one corner that is visible in atomic forcemicroscope (AFM. FIGS. 2, A and B) and transmission electron microscope(TEM) images (FIGS. 2, C and D). Gridiron structures with 63-by-63-bpcavities (FIGS. 2, E and F) were assembled to demonstrate theprogrammability of the design strategy.

To test whether the ssDNA scaffold is required to force the junction torotate and form the intended gridiron structures, we designed andsuccessfully constructed a scaffold-free 11-by-Il gridiron structure. Wealso found that scaffolded and scaffold-free gridiron elements can becombined within a single structure. Further, a scaffold-free gridironunit was examined by native gel electrophoresis to verify its formationwhen the component strands were mixed in equal stoichiometric ratios.Although the schematic diagram in FIG. 1D depicts 90° angles between thehelices in the upper and lower layers, the angles are not fixed becausethe junctions are flexible. The experimental results reveal theformation of rhomboid rather than square structures; the junctions mostlikely behave cooperatively in order to maintain optimized base-stackinginteractions and the lowest overall free energy. The single-strandedscaffold loop in one corner serves as an intrinsic marker to determinethe angles adopted by the gridiron, and the angles display a bimodaldistribution with nearly equal amplitudes, centered at 76 T 7° (SD) and103° T 70.

The flexibility of the joints makes it possible to control orreconfigure the conformation of the gridiron structure by exertingexternal forces on selected corners of a gridiron. A modified version ofa 15-by-15 gridiron structure with 21-bp cavities has about one quadrantof the gridiron unfolded and forms a randomly coiled 836-ntsingle-stranded loop between two “arms” of tweezers (FIG. 2G). The ssDNAloop is long enough to allow the structure to adopt a relaxedconformation. The observed distribution of the inner angle (q) of thetweezers (measured from 309 individual structures) is broad and centeredat 80° to 90°.

We could contract and extend the ssDNA loop by introducing secondary ortertiary structures that generate enough force to control the angle.Sets of staple strands were designed to either contract the ssDNA loopand fix an acute angle (a narrow distribution centered at 41° T 7°) viathe formation of a two-helix bundle (FIG. 2H) or to extend the loop tosecure a right (FIG. 2I) or obtuse angle (FIG. 2J) via the formation ofa three-helix bundle of specific length. The design with the right angleshows a narrow and symmetrical distribution centered at 94° T 10°, andthe design with the obtuse angle has a broader angle distributioncentered at 102 and exhibits an asymmetry that is more heavily weightedtoward smaller angles.

We extended the gridiron design into the third dimension by threedifferent strategies. The first involves stacking multiple layers of 2Dgridiron lattices at selected connection points (FIGS. 3, A and B). Thesecond relies on intertwining several gridiron planes in x-y-zdirections (FIG. 3C). The third method has its basis in distorting asingle layer of DNA gridiron into 3D structures by controlling theircurvatures (FIG. 4). By using the first strategy, we constructed athree-layer hexagonal (FIG. 3D), a four-layer rectangular gridiron (FIG.3E), and a three-layer parallelogram structure. For all multilayergridiron structures, the scaffold strand raster fills each layer, withan off-set in the angle formed between the helices of adjacent layers.The three-layer hexagonal and four-layer rectangular structuresmaintained 60° and 90° offsets between layers, respectively.

Varying the location and distance between connection points will yielddifferently patterned multilayer structures. In contrast to the angleflexibility present in the quasi-2D structures, the addition of a thirdlayer fixes the angles at junction points. The only exception to this isfor connections through the center of the same unit motif, as shown bythe green dashed line (FIG. 3A). In a 3D model of aneight-by-eight-by-eight three-layer hexagonal gridiron structure (FIG.3D), neighboring junctions in the top and bottom layers are 52 bp apart,and neighboring junctions in the middle layer (alternating connectionsto the top and bottom layers) are 26 bp apart. Because X=Y=L (FIG. 3B),each junction should adopt a 60° torsion angle. A four-layer rectangulargridiron structure (FIG. 3E) can be broken down into two six-by-fivedouble-layer gridirons (with 52-bp cavities) stacked on top of oneanother with a 26-bp offset in the connections between the first andthird, and second and fourth, layers.

The relations of the lattice planes in gridiron structures are notrestricted to stacked multilayer structures. The 3D gridiron structurescan also be assembled by integrating gridiron lattices withscaffold-free elements. FIG. 3F presents such a design in which anine-by-nine gridiron plane (shown in blue) is intertwined with aneight-by-eight scaffold-free gridiron plane (shown in yellow). Thecomplex, interwoven topology of this particular structure requiredcombining scaffolded and scaffold-free components.

Gridiron designs can allow assembly of even more complex structures byinducing a desired curvature in the basic structural unit described innonparallel helices. The relation between adjacent linear helices (theangles formed by their theoretical intersection) between adjacent linearhelices was varied. Some 3D gridiron structures that contain curvaturewere also achieved, such as the sphere shown in FIG. 4B. The helices inconcentric ring and radial spoke layers are stretched in the center andshrunk at the edges, forming a latitudinal and longitudinal framework,respectively. This is realized by progressively adjusting the distancebetween junctions in latitudinal directions. Additional modifications tothe basic structural motif can be used to produce other complexstructures. In the screw structure (FIG. 4C), the polarity of the DNAstrands in the square unit motif differs from what is illustrated inFIG. 1B (where adjacent scaffold helices have an antiparallel polarityin one direction and the same polarity in the other direction). Thescaffold strand is arranged in an antiparallel configuration to form awireframe cylinder structure (I helices are arranged axially) andsubsequently wraps around the cylinder (analogous to a left-handedscrew) until the two ends meet. The distance between adjacent axialhelices is 21 bp, the interthread distance is 42 bp, and the AFM and TEMimages display the expected left-handed conformation.

The design principles of creating gridiron units allow scaffold strandsto travel in multiple directions, which represent an important departurefrom certain aspects of the previous DNA origami methods. TraditionalHolliday junctions do not naturally adopt conformations that would allowthem to be connected in such a way, and it was unexpected to find thatthese motifs could (within a larger network of crossovers) endure a 150°rotation of one of the arms while simultaneously maintaining theirintegrity. Indeed, the flexible and dynamic behavior of these motifs mayhave excluded these types of junction conformations for consideration inscaffolded structures. Yield analysis from agarose gel and TEM imagesshows that the structures, even without purification, form withreasonably high yield (from ˜36% for the gridiron tweezers to ˜85% forthe gridiron screw, estimated from agarose gels; from ˜51% for thegridiron sphere to ˜89% for the four-layer gridiron, estimated from TEMimages; see supplementary materials for yield analysis). The ability toengineer DNA gridirons that are analogous to vector-based objects, wherea series of points with defined positions in 3D space are connected bylines, is an important milestone in the development of synthetic nucleicacid structures. In particular, this opens up new opportunities toimplement the design of complex wireframe structures that are amenableto dynamic controls. A future challenge in DNA origami is to achievetrue folding, starting from a 2D sheet (miura ori), rather than the 1DM13 scaffolds commonly used in traditional DNA origami construction. Theloose 2D networks and freely rotating hinges between different planes ofDNA gridirons provide the design features necessary to implement Miuraon type of origami.

EXAMPLES

It should be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these following Examples, one skilled inthe art can ascertain the essential characteristics of this invention,and without departing from the spirit and scope thereof, can makevarious changes and modifications of the invention to adapt it tovarious uses and conditions.

Materials and Methods

All staple strands were purchased from Integrated DNA Technologies Inc.(www.IDTDNA.com) in the format of 96-well plates at a 25 nmole synthesisscale. All the strands were normalized to 200 μM×100 μL and were usedwithout further purification. M13mp18 single stranded DNA was purchasedfrom New England Biolabs (NEB, Catalog number: #N4040S) and was used asreceived.

Assembly of 2D and 3D DNA nanostructures. The design and sequences ofthe DNA oligos used to form a particular structure are listed below. Foreach design, 10 nM of single stranded M13mp18 DNA (7,249 nucleotides)was mixed with a 10 times molar excess of staple strands in TAE Mg2+buffer (40 mM Tris. 20 mM Acetic acid, 2 mM EDTA and 12.5 mM Magnesiumacetate, pH 8.0). The resulting solutions were annealed from 95° C. to4° C. to form the designed structures. The exact temperature steps forthe slow anneal are as follows: 94 to 86° C. at 4° C. per 5 minutes; 85to 70° C. at 1° C. per 5 minutes; 70 to 40° C. at 1° C. per 15 minutes;40 to 25° C. at 1° C. per 10 minutes. The exact temperature steps forthe fast anneal are as follows: 90 to 76° C. at 2° C. per 5 minutes; 76to 24° C. at 4° C. per 5 minutes. All structures form in both annealprotocols. All samples are then subjected to AFM imaging and TEM imagingwithout further purification.

AFM imaging. For AFM imaging, the sample (2 L) was deposited onto afreshly cleaved mica surface (Ted Pella, Inc.) and left to adsorb for 2min. 50 L buffer (1×TAE-Mg2+, plus 2 L 100 mM NiCl2) was added onto themica, and the sample was scanned on a Veeco 8 AFM in the Scanasyst inFluid mode using scanasyst in fluid+ tips (Veeco, Inc.).

TEM imaging: TEM samples were prepared by dropping 2 μL of the samplesolution on a carbon-coated grid (400 mesh, Ted Pella). Beforedepositing the sample, the grids were negatively glow discharged(Emitech K100X). After 1 minute, the excess sample was wicked away fromthe grid with a piece of filter paper. To remove the excess salt, thegrid was washed with a drop of nanopure water and the excess water waswicked away with filter paper. For staining, the grid was treated with adrop of 0.7% uranyl formate solution and the excess solution was removedwith filter paper. The grid was treated with a second drop of uranylformate solution for 20 seconds, and the excess solution was removedwith filter paper. The grid was subsequently held at room temperature inair to evaporate the excess solution. TEM studies were conducted with aPhilips CM12 transmission electron microscope, operated at 80 kV inbright field mode.

Agarose Gel electrophoresis: The folding products were subject to nativegel electrophoresis on 0.75% agarose gel (1×TAE-Mg2+, preloaded in thegel with 0.5 μg/mL ethidium bromide) at 75-80 V for two to three hoursand the gels were visualized under UV light.

Page Gel electrophoresis: The folding products were subject to nativegel electrophoresis on 6% Native PAGE gel (polyacrymide; 1×TAE-Mg2+) at200V for 2 hours at 20 degree and the gels were visualized under UVlight.

Design details and sequences of assembled structures. “Tiamat” softwarewas used to design all DNA Gridiron structures. Tiamat is a basic DNAdrawing software program (similar programs also exist) and no specialalgorithms were used to design the DNA Gridiron structures. Most of thedesign tasks were performed manually and Tiamat was primarily used togenerate staple strands sequences according to the scaffold strandsequence.

FIG. 5 illustrates the design details and staple strand sequences ofsome example DNA Gridiron structures. Tiamat software and files for alldesigns are available for downloading at the following website:

TABLE 1 Sequences of the staples in the 21 bps Gridiron structureName (No.) Sequence 21bpsGridiron-1GAAAATTCATAAGTAAGCGTCATACATGGCTTAGACGGGAGA (SEQ ID NO. 1)21bpsGridiron-2ATATTCACAAAATAAAAACAGGGAAGCGCATTTTGATGATAC (SEQ ID NO. 2)21bpsGridiron-3AGGAGTGTACTAATAAACAGCCATATTATTTAATTGGCCTTG (SEQ ID NO. 3)21bpsGridiron-4GCCAGTTACAAGGTAATAAGTTTTAACGGGGTGTCCTGAACA (SEQ ID NO. 4)21bpsGridiron-5GCAGAACGCGCGAGGTTGAGGCAGGTCAGACGTCCCAATCCA (SEQ ID NO. 5)21bpsGridiren-6AATAAGAAACGGACTTGAGCCATTTGGGAATTTTCAGCTAAT (SEQ ID NO. 6)21bpsGridiron-7GAGAGAATAACCAAATAAATCCTCATTAAAGCAAAAGGGCGA (SEQ ID NO. 7)21bpsGridiron-8AGCATTGACAGCTGTTTATCAACAATAGATAACAGTGCCTTG (SEQ ID NO. 8)21bpsGridiron-9AGTAACAGTGCCCAGTAATAAGAGAATATAAAAGCCGCCGCC (SEQ ID NO. 9)21bpsGridiron-10GCATTTTCGAGCCGTATAAACAGTTAATGCCCTATCAAAATC (SEQ ID NO. 10)21bpsGridiron-11ATTAAGACGCTCGCCACCAGAACCACCACCAGGTACCGACAA (SEQ ID NO. 11)21bpsGridiren-12AAGGTAAAGTAAGCACCATTACCATTAGCAAGGATAGCTTAG (SEQ ID NO. 12)21bpsGridiron-13TTTACCGTTCCTGGTTTACCAGCGCCAAAGACCAGAATGGAAAG (SEQ ID NO. 13)21bpsGridiron-14CATTCAACCGATTGACGGAAATTATTCATTAAAGCCTTTACA (SEQ ID NO. 14)21bpsGridiron-15GAAAATAGCAGGTGAATTATCACCGTCACCATTTTTTGTT (SEQ ID NO. 15)21bpsGridiron-16GCAAAGACACCGTAAATGAATTTTCTGTATGGTAATTGAGCG (SEQ ID NO. 16)21bpsGridiron-17TAGCATTCCACACCCTGAACAAAGTCAGAGGGGATTTTGCTA (SEQ ID NO. 17)21bpsGridiron-18AACAACTTTCACGCTAACGAGCGTCTTTCCAGACAACGCCTG (SEQ ID NO. 18)21bpsGridiren-19AATCTTACCAAACAGTTTCAGCGGAGTGAGAAATGTAGAAAC (SEQ ID NO. 19)21bpsGridiron-20AGAAAAATAATGTTTCGTCACCAGTACAAACTAGCCTAATTT (SEQ ID NO. 20)21bpsGridiron-21ATTAACTGAACAGACAGCCCTCATAGTTAGCGACAATCAATA (SEQ ID NO. 21)21bpsGridiron-22AACAACATGAGAGCCAGCAAAATCACCAGTATTCTGTCCA (SEQ ID NO. 22)21bpsGridiron-23CGTAACACTGAATCCCATCCTAATTTACGAGCTAGAAAGGAA (SEQ ID NO. 23)21bpsGridiron-24CAACTAAAGGATTAACAACGCCAACATGTAATACCCATGTAC (SEQ ID NO. 24)21bpsGridiron-25AATCGCCATATATTGCGAATAATAATTTTTTCGCTTAGGTTG (SEQ ID NO. 25)21bpsGridiren-26ATAGGTCTGAGGGGATAGCAAGCCCAATAGGATTAGGCAGAG (SEQ ID NO. 26)21bpsGridiron-27TCCAGACGTTAACGGAATAAGTTTATTTTGTCTAACGATCTAAA (SEQ ID NO. 27)21bpsGridiron-28TCGCCCACGCAGCCATTGCAACAGGAAAAATGCGCCGACA (SEQ ID NO. 28)21bpsGridiron-29CCTCATTTTCAAGACTACCTTTTTAACCTCCGACGTTGAAAA (SEQ ID NO. 29)21bpsGridiron-30TCTCCAAAAAATGAATTACCTTTTTTAATGGAGAGCCACCAC (SEQ ID NO. 30)21bpsGridiron-31ATATAAGTATTTGACGCTCAATCGTCTGAAGATAAGTGCC (SEQ ID NO. 31)21bpsGridiren-32ACCCTCAGAGCGAGAAGAGTCAATAGTGAATTCCTGCCTATT (SEQ ID NO. 32)21bpsGridiron-33TCGGAACCTATTGTGAGTGAATAACCTTGCTTCAGAGCCACC (SEQ ID NO. 33)21bpsGridiron-34CGTTTGCCAATTCACCAGTCACACGACCAGTTCGGTCATA (SEQ ID NO. 34)21bpsGridiron-35AAACATAGCGCCGGAAACGTCACCAATGAATAATTTTCCC (SEQ ID NO. 35)21bpsGridiron-36ACAATTTCATTAAGGCTCCAAAAGGAGCCTTTTATACTTCTG (SEQ ID NO. 36)21bpsGridiron-37GAACAAAGAATCAGTAGCGACAGAATCAAGTTGAGTAACA (SEQ ID NO. 37)21bpsGridiron-38GATAATACATTTGCTTTCGAGGTGAATTTCTTAGCCCTAAAA (SEQ ID NO. 38)21bpsGridiren-39ACAGAGATATAGCGCGTTTTCATCGGCATTTAATAAAAGG (SEQ ID NO. 39)21bpsGridiron-40ATTTTAAAAGTTTTGCCTTTAGCGTCAGACTGGAACCCTTCT (SEQ ID NO. 40)21bpsGridiron-41GACCTGAAAGCCGGAACCAGAGCCACCACCGGAACGTTATTA (SEQ ID NO. 41)21bpsGridiron-42ATCAAAATCACGTAAGAATACGTGGCACAGACGGTTTTGCTC (SEQ ID NO. 42)21bpsGridiron-43AGTACCAGGCGATGGATTATTTACATTGGCAGTOTTTTCATA (SEQ ID NO. 43)21bpsGridiron-44AATTCGACAACGAGAAGGATTAGGATTAGCGGAATATTTTTG (SEQ ID NO. 44)21bpsGridiron-45AATGGCTATTACCGTACTCAGGAGGTTTAGTACTTTACAAAC (SEQ ID NO. 45)21bpsGridiron-46TAGGTGTATCAGTCTTTAATGCGCGAACTGATAAACAGCTTG (SEQ ID NO. 46)21bpsGridiron-47ATACCGATAGTCGCTCATGGAAATACCTACATTAGCCCGGAA (SEQ ID NO. 47)21bpsGridiron-48GTTTATCAGCTTGAGGATTTAGAAGTATTAGACCGCCACCCT (SEQ ID NO. 48)21bpsGridiron-49CAGAACCGCCATATAATCCTGATTGTTTGGATAATTGTATCG (SEQ ID NO. 49)21bpsGridiron-50AGACTCCTCAATCGTATTAAATCCTTTGCCCGAACCGCCTCC (SEQ ID NO. 50)21bpsGridiron-51CTCAGAGCCGCTATCATCATATTCCTGATTATTAAGAGGCTG (SEQ ID NO. 51)21bpsGridiron-52TCGCTATTAATACCATCGATAGCAGCACCGTAAACCACCAGA (SEQ ID NO. 52)21bpsGridiron-53AGGAGCGGAATCACCCTCAGAACCGCCACCCTCTGTAAATCG (SEQ ID NO. 53)21bpsGridiron-54AAATCAATATATATTCTGAAACATGAAAGTATCAGATGATGG (SEQ ID NO. 54)21bpsGridiron-55CAATTCATCAACCCTCAGAACCGCCACCCTCAAACAGTACAT (SEQ ID NO. 55)21bpsGridiron-56GGTTATATAACCGGCTACAGAGGCTTTGAGGACTTAATTGAG (SEQ ID NO. 56)21bpsGridiron-57TAAGGCGTTCCCAATTCTGCGAACGAGTAGTGAAATACCG (SEQ ID NO. 57)21bpsGridiron-58GCTTAATTGCTAACGCAATAATAACGGAATACAGGTCATTTTTG (SEQ ID NO. 58)21bpsGridiron-59TAATTTCATCTACTTCAAATATCGCGTTTTAATCATAATTAC (SEQ ID NO. 59)21bpsGridiron-60TAGAAAAAGCCGTTTACCAGACGACGATAAAAATATTTTAGT (SEQ ID NO. 60)21bpsGridiron-61AGTTGAGATTTAGACTCCTTATTACGCAGTATATTATTACAGGT (SEQ ID NO. 61)21bpsGridiron-62AAGACAAAGAATAATCATTGTGAATTACCTTATACAAATTCT (SEQ ID NO. 62)21bpsGridiron-63TACCAGTATAATCCATGTTACTTAGCCGGAACATCCAATCGC (SEQ ID NO. 63)21bpsGridiron-64ATCTTTGACCCATACATAAAGGTGGCAACATAGGCAAAAGAATA (SEQ ID NO. 64)21bpsGridiron-65AACCGAGGAGAATATAATGCTGTAGCTCAAGCCGAACAAA (SEQ ID NO. 65)21bpsGridiron-66CTAATATCAGAGCACCAACCTAAAACGAAAGATAAAAGAAAC (SEQ ID NO. 66)21bpsGridiron-67CCACTACGAAGGAGATAACCCACAAGAATTGAAAACAAAGTA (SEQ ID NO. 67)21bpsGridiron-68CAACGGAGATTCTATTTTGCACCCAGCTACAAATACGTAATG (SEQ ID NO. 68)21bpsGridiron-69TAGAAGGCTAAGTACGGTGTCTGGAAGTTTCCCAATAGCA (SEQ ID NO. 69)21bpsGridiron-70CAATCAATAATAGTTTCCATTAAACGGGTAAATTTTATCCTG (SEQ ID NO. 70)21bpsGridiron-71TTTCATGAGGACGGCTGTCTTTCCTTATCATTGTGTCGAAAT (SEQ ID NO. 71)21bpsGridiron-72CCGCGACCTGCAGCCAACGCTCAACAGTAGGGCTAAAGACTT (SEQ ID NO. 72)21bpsGridiron-73AAGATTAGTTGTGTATCATCGCCTGATAAATTCCAAGAACGG (SEQ ID NO. 73)21bpsGridiron-74GTATTAAACCAATTATACCAGTCAGGACGTTGGCCTTAAATC (SEQ ID NO. 74)21bpsGridiron-75AGAACTGGCTCAGTACCGCACTCATCGAGAACAGCAACACTA (SEQ ID NO. 75)21bpsGridiron-76TCATAACCCTCTGTTTAGTATCATATGCGTTATGCGATTTTA (SEQ ID NO. 76)21bpsGridiron-77AGCGAACCTCCGGAATTACGAGGCATAGTAAGAAGCAAGCCG (SEQ ID NO. 77)21bpsGridiron-78TTTTTATTTTCGACCGGAAGCAAACTCCAACAGAGGCGTTTT (SEQ ID NO. 78)21bpsGridiron-79AAAGCGAACCAATCGTAGGAATCATTACCGCGCATTCCATAT (SEQ ID NO. 79)21bpsGridiron-80AACAGTTGATTAAATAAGAATAAACACCGGAATTCGAGCTIC (SEQ ID NO. 80)21bpsGridiron-81ATATGCAACTATATCCGGTATTCTAAGAACGCGGTCAGGATT (SEQ ID NO. 81)21bpsGridiron-82AGAGAGTACCTTTTAAGAAAAGTAAGCAGATACATGTTTTAA (SEQ ID NO. 82)21bpsGridiron-83TAACGCCAAAACGACTTGCGGGAGGTTTTGAAGGAAGAAAAA (SEQ ID NO. 83)21bpsGridiron-84TCTACGTTAATAAGAAACAATGAAATAGCAATTGCAGATACA (SEQ ID NO. 84)21bpsGridiron-85GTAGAAAATACCCAGCGATTATACCAAGCGCGGTTAAGCCCA (SEQ ID NO. 85)21bpsGridiron-86ATAATAAGAGCAAAACGAACTAACGGAACAACGTTAGCAAAC (SEQ ID NO. 86)21bpsGridiron-87TGGCATGATTAAGGAATACCACATTCAACTAAAGCTATCTTA (SEQ ID NO. 87)21bpsGridiron-88CCGAAGCCCTTTTAATTGCTCCTTTTGATAAGCCAAAAGAAC (SEQ ID NO. 88)21bpsGridiron-89AAGCCCGAAAGTCTGACCTAAATTTAATGGTTATTTAGTTTG (SEQ ID NO. 89)21bpsGridiron-90ACCATTAGATAGATTGCTTTGAATACCAAGTTGATTAAGAGG (SEQ ID NO. 90)21bpsGridiron-91TAAACAGTTTTTGATTAGTAATAACATCACCATTGAATCC (SEQ ID NO. 91)21bpsGridiron-92AATTTCAACTTCGCGAGAAAACTTTTTCAAATACCAAAATAG (SEQ ID NO. 92)21bpsGridiron-93CGAGAGGCTTTTTATTCATTTCAATTACCTGAGAGATGGTTT (SEQ ID NO. 93)21bpsGridiron-94ATTCATTACAACTATCGGCCTTGCTGGTAAAGTAATCTTG (SEQ ID NO. 94)21bpsGridiron-95GAGGGTAGCAATATATGTAAATGCTGATGCAAGAGGCGCAGA (SEQ ID NO. 95)21bpsGridiron-96CGGTCAATCATAACATCAAGAAAACAAAATTAGCATCGGAAC (SEQ ID NO. 96)21bpsGridiron-97GATTCGCCTCATTTCGCAAATGGTCAATAATTACATCGGG (SEQ ID NO. 97)21bpsGridiron-98AATAATGGAAGCACCCTCAGCAGCGAAAGACAATTACATTTA (SEQ ID NO. 98)21bpsGridiron-99TGCGGGATCGTGGTTAGAACCTACCATATCAATTTGAAAGAG (SEQ ID NO. 99)21bpsGridiron-100GACAGATGAACACTAACAACTAATAGATTAGAAGGCCGCTTT (SEQ ID NO. 100)21bpsGridiron-101CTCAAATATTTGGGGCGCGAGCTGAAAAGGTCTAAAGCAT (SEQ ID NO. 101)21bpsGridiron-102CATCGCCATTACTGAGGCTTGCAGGGAGTTAAGCCGTCAATA (SEQ ID NO. 102)21bpsGridiron-103ATATTCGGTCGAAAATACCGAACGAACCACCAGGCTGGCTGA (SEQ ID NO. 103)21bpsGridiron-104CCTTCATCAAGTATCCAGAACAATATTACCGCCATAACCGAT (SEQ ID NO. 104)21bpsGridiron-105TCTTTAGGAGCGGTGTACAGACCAGGCGCATAGCAGAAGATA (SEQ ID NO. 105)21bpsGridiron-106AAACAGAGGTGGCTCATTCAGTGAATAAGGCTATCTAAAATA (SEQ ID NO. 106)21bpsGridiron-107GTAACAAAGCTAGGCGGTCAGTATTAACACCGTGCGGAATCG (SEQ ID NO. 107)21bpsGridiron-108TCATAAATATTTTGCCTGAGTAGAAGAACTCACCAAATCAAC (SEQ ID NO. 108)21bpsGridiron-109AAATCAACAGTAGACTGGATAGCGTCCAATACCCTGCAACAG (SEQ ID NO. 109)21bpsGridiron-110TGCCACGCTGAAATCAAAAATCAGGTCTTTACGTCAGTTGGC (SEQ ID NO. 110)21bpsGridiron-111GAATGACCATAGAGCCAGCAGCAAATGAAAAATGGCATCAAT (SEQ ID NO. 111)21bpsGridiron-112TCTACTAATAGTAACCGTTGTAGCAATACTTCCAGAAAACGA (SEQ ID NO. 112)21bpsGridiron-113TATATTTTCATCAAACCCTCAATCAATATCTGCCTGACTATT (SEQ ID NO. 113)21bpsGridiron-114ATAGTCAGAAGAATATACAGTAACAGTACCTTCCTGTTTAGC (SEQ ID NO. 114)21bpsGridiron-115GTAAAATGTTTTGAAAGGAATTGAGGAAGGTTTGCCCTGACG (SEQ ID NO. 115)21bpsGridiron-116AGAAACACCAGAAATAAAGAAATTGCGTAGATGGGGGTAATA (SEQ ID NO. 116)21bpsGridiron-117ATGATGAAACAAAGGGAACCGAACTGACCAACAATTATTTGC (SEQ ID NO. 117)21bpsGridiron-118ACGTAAAACAGAACGAGTAGTAAATTGGGCTTGCAAAAGAAG (SEQ ID NO. 118)21bpsGridiron-119GCAGAGGCGAATGCAAAAGAAGTTTTGCCAGATTTCAGGTTT (SEQ ID NO. 119)21bpsGridiron-120AACGTCAGATGCAAAGCGGATTGCATCAAAAAACAAAATCGC (SEQ ID NO. 120)

TABLE 2 Sequences of the staples in the 42 bps Gridiron structureName (No.) Sequence 42bpsGridiron-1CCTCCCGACTTGCGGGAGGTTCTGCATTAATGAATCGGCCAA (SEQ ID NO. 121)42bpsGridiron-2TAACTCACATTAATTGCGTTGAGAATTAACTGAACACCCTGA (SEQ ID NO. 122)42bpsGridiron-3AAAATGAAAATAGCAGCCTTTTTAAATTTTTCTTAAATCAGC (SEQ ID NO. 123)42bpsGridiron-4AACAGGAAGATTGTATAAGCATACAATTTTATCCTGAATCTT (SEQ ID NO. 124)42bpsGridiron-5AGTTGCTATTTTGCACCCAGCAATATTTAAATTGTAAACGTT (SEQ ID NO. 125)42bpsGridiron-6AATATTTTGTTAAAATTCGCAACAGAGAGAATAACATAAAAA (SEQ ID NO. 126)42bpsGridiron-7CAGGGAAGCGCATTAGACGGGCGCTCACTGCCCGCTTTCCAG (SEQ ID NO. 127)42bpsGridiron-8TCGGGAAACCTGTCGTGCCAGTTGAAGCCTTAAATCAAGATT (SEQ ID NO. 128)42bpsGridiron-9ACCAACGCTAACGAGCGTCTTTGTCAATCATATGTACCCCGG (SEQ ID NO. 129)42bpsGridiron-10GGTCATTGCCTGAGAGTCTGGACGATTTTTTCTTTAACGTCA (SEQ ID NO. 130)42bpsGridiron-11TTATCCCAATCCAAATAAGAAAGCAAACAAGAGAATCGATGA (SEQ ID NO. 131)42bpsGridiron-12ACGGTAATCGTAAAACTAGCATCCAGAGCCTAATTTGCCAGT (SEQ ID NO. 132)42bpsGridiron-13CCGCCACCCTCAGAGCCACCATTTCATCAACATTAAATGTGA (SEQ ID NO. 133)42bpsGridiron-14TCATTTTTTAACCAATAGGAAGTAGCGCGTTTTCATCGGCAT (SEQ ID NO. 134)42bpsGridiron-15AACCATCGATAGCAGCACCGTTGGGGTGCCTAATGAGTGAGC (SEQ ID NO. 135)42bpsGridiron-16AGCTTGCATGCCTGCAGGTCGTAGTTGCGCCGACAATGACAA (SEQ ID NO. 136)42bpsGridiron-17TTTCGGTCATAGCCCCCTTATAGAGATCTACAAAGGCTATCA (SEQ ID NO. 137)42bpsGridiron-18CCTCATATATTTTAAATGCAAAAAAAAGGCTCCAAAAGGAGC (SEQ ID NO. 138)42bpsGridiron-19TTTCACGTTGAAAATCTCCAATGCCTGAGTAATGTGTAGGTA (SEQ ID NO. 139)42bpsGridiron-20AAGATTCAAAAGGGTGAGAAATGAGAATAGAAAGGAACAACT (SEQ ID NO. 140)42bpsGridiron-21TCATAGTTAGCGTAACGATCTTGGTCATAGCTGTTTCCTGTG (SEQ ID NO. 141)42bpsGridiron-22CCGAGCTCGAATTCGTAATCAAAAGTTTTGTCGTCTTTCCAG (SEQ ID NO. 142)42bpsGridiron-23ACGTTAGTAAATGAATTTTCTTCTCCGTGGGAACAAACGGCG (SEQ ID NO. 143)42bpsGridiron-24GCGAGTAACAACCCGTCGGATGTATGGGATTTTGCTAAACAA (SEQ ID NO. 144)42bpsGridiron-25CTTTAATTGTATCGGTTTATCTCACGTTGGTGTAGATGGGCG (SEQ ID NO. 145)42bpsGridiron-26GATTGACCGTAATGGGATAGGAGCTTGCTTTCGAGGTGAATT (SEQ ID NO. 146)42bpsGridiron-27CTTTCAACAGTTTCAGCGGAGGGCCGGAGACAGTCAAATCAC (SEQ ID NO. 147)42bpsGridiron-28CATCAATATGATATTCAACCGTCAGAGCCGCCACCCTCAGAA (SEQ ID NO. 148)42bpsGridiron-29CCACCACCGGAACCGCCTCCCTTCTAGCTGATAAATTAATGC (SEQ ID NO. 149)42bpsGridiron-30TGAAATTGTTATCCGCTCACAGCATTGACAGGAGGTTGAGGC (SEQ ID NO. 150)42bpsGridiron-31CCACCACCAGAGCCGCCGCCAATTCCACACAACATACGAGCC (SEQ ID NO. 151)42bpsGridiron-32TCTGGCCTTCCTGTAGCCAGCCCCTCAGAGCCGCCACCAGAA (SEQ ID NO. 152)42bpsGridiron-33CGGAGAGGGTAGCTATTTTTGTAGCGTTTGCCATCTTTTCAT (SEQ ID NO. 153)42bpsGridiron-34TCTTAAACAGCTTGATACCGAACTCTAGAGGATCCCCGGGTA (SEQ ID NO. 154)42bpsGridiron-35GGAAGCATAAAGTGTAAAGCCAATCAGTAGCGACAGAATCAA (SEQ ID NO. 155)42bpsGridiron-36GTTTGCCTTTAGCGTCAGACTCGCCATCAAAAATAATTCGCG (SEQ ID NO. 156)42bpsGridiron-37ACAGGTAGAAAGATTCATCAGACTCCAGCCAGCTTTCCGGCA (SEQ ID NO. 157)42bpsGridiron-38CATCGTAACCGTGCATCTGCCTGGTTTAATTTCAACTTTAAT (SEQ ID NO. 158)42bpsGridiron-39ATTCAGTGAATAAGGCTTGCCGTAAAACGACGGCCAGTGCCA (SEQ ID NO. 159)42bpsGridiron-40CATTGTGAATTACCTTATGCGAAGGATAAAAATTTTTAGAAC (SEQ ID NO. 160)42bpsGridiron-41TAGCALAATTAAGCAATAAAGTCTACTAATAGTAGTAGCATT (SEQ ID NO. 161)42bpsGridiron-42CGAACGAGTAGATTTAGTTTGCGCTATTACGCCAGCTGGCGA (SEQ ID NO. 162)42bpsGridiron-43GGCGATCGGTGCGGGCCTCTTACCATTAGATACATTTCGCAA (SEQ ID NO. 163)42bpsGridiron-44ATGGTCAATAACCTGTTTAGCAGGCAAAGCGCCATTCGCCAT (SEQ ID NO. 164)42bpsGridiron-45CCGCTTCTGGTGCCGGAAACCTATATTTTCATTTGGGGCGCG (SEQ ID NO. 165)42bpsGridiron-46AGCTGAAAAGGTGGCATCAATCCTCAGAGCATAAAGCTAAAT (SEQ ID NO. 166)42bpsGridiron-47CGGTTGTACCAAAAACATTATAACTAACGGAACAACATTATT (SEQ ID NO. 167)42bpsGridiron-48AAAATCTACGTTAATAAAACGGACCCTGTAATACTTTTGCGG (SEQ ID NO. 168)42bpsGridiron-49AAGGGGGATGTGCTGCAAGGCACGCCAAAAGGAATTACGAGG (SEQ ID NO. 169)42bpsGridiron-50TTCAACTAATGCAGATACATAGATTAAGTTGGGTAACGCCAG (SEQ ID NO. 170)42bpsGridiron-51TATCGGCCTCAGGAAGATCGCTTGAGATTTAGGAATACCACA (SEQ ID NO. 171)42bpsGridiron-52GAGAAGCCTTTATTTCAACGCATTTTAAGAACTGGCTCATTA (SEQ ID NO. 172)42bpsGridiron-53GGTTTTCCCAGTCACGACGTTCTGACGAGAAACACCAGAACG (SEQ ID NO. 173)42bpsGridiron-54AGTAGTAAATTGGGCTTGAGAAGTTTGAGGGGACGACGACAG (SEQ ID NO. 174)42bpsGridiron-55TCTTTCCTTATCATTCCAAGACGTAAAACAGAAATAAAGAAA (SEQ ID NO. 175)42bpsGridiron-56TTGTTTGGATTATACTTCTGAAAAGTTACCAGAAGGAAACCG (SEQ ID NO. 176)42bpsGridiron-57AATGAAATAGCAATAGCTATCAATGGATTATTTACATTGGCA (SEQ ID NO. 177)42bpsGridiron-58CCAGCCATTGCAACAGGAAAAGCCGTTTTTATTTTCATCGTA (SEQ ID NO. 178)42bpsGridiron-59GCACTCATCGAGAACAAGCAAACGCTCATGGAAATACCTACA (SEQ ID NO. 179)42bpsGridiron-60TTTTGACGCTCAATCGTCTGATTACCGAAGCCCTTTTTAAGA (SEQ ID NO. 180)42bpsGridiron-61AAAGTAAGCAGATAGCCGAACATAATGGAAGGGTTAGAACCT (SEQ ID NO. 181)42bpsGridiron-62ACCATATCAAAATTATTTGCAACGGGTATTAAACCAAGTACC (SEQ ID NO. 182)42bpsGridiron-63GGAATCATTACCGCGCCCAATTCAAACTATCGGCCTTGCTGG (SEQ ID NO. 183)42bpsGridiron-64AATTAACCGTTGTAGCAATACCCAATAATAAGAGCAAGAAAC (SEQ ID NO. 184)42bpsGridiron-65ACAAAGTCAGAGGGTAATTGACCGCCTGGCCCTGAGAGAGTT (SEQ ID NO. 185)42bpsGridiron-66TATTGGGCGCCAGGGTGGTTTAACGCGAGGCGTTTTAGCGAA (SEQ ID NO. 186)42bpsGridiron-67AGGCTTATCCGGTATTCTAAGTTCTTTTCACCAGTGAGACGG (SEQ ID NO. 187)42bpsGridiron-68GCAACAGCTGATTGCCCTTCAGCGCTAATATCAGAGAGATAA (SEQ ID NO. 188)42bpsGridiron-69CCCACAAGAATTGAGTTAAGCTTCTTTGATTAGTAATAACAT (SEQ ID NO. 189)42bpsGridiron-70CACTTGCCTGAGTAGAAGAACAGCAAGCAAATCAGATATAGA (SEQ ID NO. 190)42bpsGridiron-71TTCCAGTAAGCGTCATACATGTGACCTGAAAGCGTAAGAATA (SEQ ID NO. 191)42bpsGridiron-72GATTCACCAGTCACACGACCAAAGGTGAATTATCACCGTCAC (SEQ ID NO. 192)42bpsGridiron-73CAAAAGGGCGACATTCAACCGAATTCATCAATATAATCCTGA (SEQ ID NO. 193)42bpsGridiron-74TTTACAAACAATTCGACAACTACTTTTTCATGAGGAAGTTTC (SEQ ID NO. 194)42bpsGridiron-75CAACCATCGCCCACGCATAACAAAGAACGTGGACTCCAACGT (SEQ ID NO. 195)42bpsGridiron-76GCAGCAAGCGGTCCACGCTGGGGCCGGAAACGTCACCAATGA (SEQ ID NO. 196)42bpsGridiron-77CGACTTGAGCCATTTGGGAATAAAGAGTCTGTCCATCACGCA (SEQ ID NO. 197)42bpsGridiron-78TGGTTGCTTTGACGAGCACGTCTTTTGCGGGATCGTCACCCT (SEQ ID NO. 198)42bpsGridiron-79CTTGCAGGGAGTTAAAGGCCGATAACGTGCTTTCCTCGTTAG (SEQ ID NO. 199)42bpsGridiron-80AATCAGAGCGGGAGCTAAACACCGTAACACTGAGTTTCGTCA (SEQ ID NO. 200)42bpsGridiron-81GGAGGTTTAGTACCGCCACCCTGAGTAACATTATCATTTTGC (SEQ ID NO. 201)42bpsGridiron-82ACGTTATTAATTTTAAAAGTTTCAGAACCGCCACCCTCAGAA (SEQ ID NO. 202)42bpsGridiron-83CCGCCACCCTCAGAGCCACCAGAATGGCTATTAGTCTTTAAT (SEQ ID NO. 203)42bpsGridiron-84CGTGGCACAGACAATATTTTTCCCTCATTTTCAGGGATAGCA (SEQ ID NO. 204)42bpsGridiron-85CAGCAGCGAAAGACAGCATCGACATCGCCATTAAAAATACCG (SEQ ID NO. 205)42bpsGridiron-86GCGCGAACTGATAGCCCTAAAGAACGAGGGTAGCAACGGCTA (SEQ ID NO. 206)42bpsGridiron-87AGCCCAATAGGAACCCATGTAGGAGGCCGATTAAAGGGATTT (SEQ ID NO. 207)42bpsGridiron-88ATCAAAAGAATAGCCCGAGATGTAGCATTCCACAGACAGCCC (SEQ ID NO. 208)42bpsGridiron-89CCAGTACAAACTACAACGCCTAGGGTTGAGTGTTGTTCCAGT (SEQ ID NO. 209)42bpsGridiron-90TAGACAGGAACGGTACGCCAGGCGCAGTCTCTGAATTTACCG (SEQ ID NO. 210)42bpsGridiron-91AGGTCAGACGATTGGCCTTGAAATCGGCAAAATCCCTTATAA (SEQ ID NO. 211)42bpsGridiron-92CCTGTTTGATGGTGGTTCCGATATTCACAAACAAATAAATCC (SEQ ID NO. 212)42bpsGridiron-93TCATTAAAGCCAGAATGGAAAAATCCTGAGAACTGTTTTTAT (SEQ ID NO. 213)42bpsGridiron-94GGAACAAAGAAACCACCAGAAGGGTCAGTGCCTTGAGTAACA (SEQ ID NO. 214)42bpsGridiron-95TACTGGTAATAAGTTTTAACGGGAGCGGAATTATCATCATAT (SEQ ID NO. 215)42bpsGridiron-96CCAACAGAGATAGAACCCTTCGCTTTTGATGATACAGGAGTG (SEQ ID NO. 216)42bpsGridiron-97AATCAGTGAGGCCACCGAGTATAGAGCCAGCAAAATCACCAG (SEQ ID NO. 217)42bpsGridiron-98TAGCACCATTACCATTAGCAATTTGCCCCAGCAGGCGAAAAT (SEQ ID NO. 218)42bpsGridiron-99TTGGAACAAGAGTCCACTATTCGATATATTCGGTCGCTGAGG (SEQ ID NO. 219)42bpsGridiron-100CAGAGGCTTTGAGGACTAAAGCGTATTAAATCCTTTGCCCGA (SEQ ID NO. 220)42bpsGridiron-101TCCTGATTATCAGATGATGGCATTGAGGGAGGGAAGGTAAAT (SEQ ID NO. 221)42bpsGridiron-102ATTGACGGAAATTATTCATTAGTAATAAAAGGGACATTCTGG (SEQ ID NO. 222)42bpsGridiron-103TTTGCCAGAGGGGGTAATAGTGTGCCACGCTGAGAGCCAGCA (SEQ ID NO. 223)42bpsGridiron-104AACGAACCACCAGCAGAAGATATGAACGGTGTACAGACCAGG (SEQ ID NO. 224)42bpsGridiron-105CGGAACGAGGCGCAGACGGTCGAGGATTTAGAAGTATTAGAC (SEQ ID NO. 225)42bpsGridiron-106CAAAGGGCGAAAAACCGTCTAATCAACGTAACAAAGCTGCTC (SEQ ID NO. 226)42bpsGridiron-107CGCATAGGCTGGCTGACCTTCGCCGCTACAGGGCGCGTACTA (SEQ ID NO. 227)42bpsGridiron-108CGTGGCGAGAAAGGAAGGGAAATATGCAACTAAAGTACGGTG (SEQ ID NO. 228)42bpsGridiron-109AGGATTAGAGAGTACCTTTAAGAAAGGAATTGAGGAAGGTTA (SEQ ID NO. 229)42bpsGridiron-110TCAGTTGGCAAATCAACAGTTTTGCTCCTTTTGATAAGAGGT (SEQ ID NO. 230)42bpsGridiron-111CATTTTTGCGGATGGCTTAGATCACCTTGCTGAACCTCAAAT (SEQ ID NO. 231)42bpsGridiron-112GCAAATGAAAAATCTAAAGCAGCTTAATTGCTGAATATAATG (SEQ ID NO. 232)42bpsGridiron-113CTGTAGCTCAACATGTTTTAAGAAAGCGAAAGGAGCGGGCGC (SEQ ID NO. 233)42bpsGridiron-114TAAAGCACTAAATCGGAACCCAACAGTTGATTCCCAATTCTG (SEQ ID NO. 234)42bpsGridiron-115TCTGGAAGTTTCATTCCATATTAAAGGGAGCCCCCGATTTAG (SEQ ID NO. 235)42bpsGridiron-116TAGGGCGCTGGCAAGTGTAGCAGAGGCTTTTGCAAAAGAAGT (SEQ ID NO. 236)42bpsGridiron-117CATAGTAAGAGCAACACTATCTTTTTTGGGGTCGAGGTGCCG (SEQ ID NO. 237)42bpsGridiron-118TGAACCATCACCCAAATCAAGATAACCCTCGTTTACCAGACG (SEQ ID NO. 238)42bpsGridiron-119ACGATAAAAACCAAAATAGCGGGTCACGCTGCGCGTAACCAC (SEQ ID NO. 239)42bpsGridiron-120TCTAAAATATCTTTAGGAGCAATAAATATTCATTGAATCCCC (SEQ ID NO. 240)42bpsGridiron-121GTCCAATACTGCGGAATCGTCCTAACAACTAATAGATTAGAG (SEQ ID NO. 241)42bpsGridiron-122GTATTAACACCGCCTGCAACAAAAATGTTTAGACTGGATAGC (SEQ ID NO. 242)42bpsGridiron-123CACACCCGCCGCGCTTAATGCATCAAGAGTAATCTTGACAAG (SEQ ID NO. 243)42bpsGridiron-124AACCGGATATTCATTACCCAATCAGGGCGATGGCCCACTACG (SEQ ID NO. 244)42bpsGridiron-125CCGTCAATAGATAATACATTTAATCATAAGGGAACCGAACTG (SEQ ID NO. 245)42bpsGridiron-126ACCAACTTTGAAAGAGGACAGAAAACAGAGGTGAGGCGGTCA (SEQ ID NO. 246)42bpsGridiron-127ATCAACAATAGATAAGTCCTGTGTCCAGACGACGACAATAAA (SEQ ID NO. 247)42bpsGridiron-128GCAGAGGCATTTTCGAGCCAGGTATGTTAGCAAACGTAGAAA (SEQ ID NO. 248)42bpsGridiron-129AGGAAACGCAATAATAACGGATTGCTTTGAATACCAAGTTAC (SEQ ID NO. 249)42bpsGridiron-130GTCAGATGAATATACAGTAACAAACCAATCAATAATCGGCTG (SEQ ID NO. 250)42bpsGridiron-131TCCTAATTTACGAGCATCTAGAGTACCTTTTACATCGGGAGA (SEQ ID NO. 251)42bpsGridiron-132AACAATAACGGATTCGCCTGAATACCCAAAAGAACTGGCATG (SEQ ID NO. 252)42bpsGridiron-133ATTAAGACTCCTTATTACGCATAATAAGAGAATATAAAGTAC (SEQ ID NO. 253)42bpsGridiron-134CGACAAAAGGTAAAGTAATTCAACAAGAAAAATAATATCCCA (SEQ ID NO. 254)42bpsGridiron-135CATTAAACGGGTAAAATACGTTGAGTGAATAACCTTGCTTCT (SEQ ID NO. 255)42bpsGridiron-136AAAATCGCGCAGAGGCGAATTATGGTTTACCAGCGCCAAAGA (SEQ ID NO. 256)42bpsGridiron-137ATAAAAGAAACGCAAAGACACCAACGCCAACATGTAATTTAG (SEQ ID NO. 257)42bpsGridiron-138GTGATAAATAAGGCCTTAAATAGAATACACTAAAACACTCAT (SEQ ID NO. 258)42bpsGridiron-139ACCTAAAACGAAAGAGGCAAAAAGAATAAACACCGGAATCAT (SEQ ID NO. 259)42bpsGridiron-140AATTACTAGAAAAAGCCTGTTGGATAAGTGCCGTCGAGAGGG (SEQ ID NO. 260)42bpsGridiron-141GGGTTTTGCTCAGTACCAGGCTAGTATCATATGCGTTATACA (SEQ ID NO. 261)42bpsGridiron-142TACATTTAACAATTTCATTTGATAGGTGTATCACCGTACTCA (SEQ ID NO. 262)42bpsGridiron-143TTGATATAAGTATAGCCCGGAAATTACCTTTTTTAATGGAAA (SEQ ID NO. 263)42bpsGridiron-144AATTCTTACCAGTATAAAGCCGTATTAAGAGGCTGAGACTCC (SEQ ID NO. 264)42bpsGridiron-145GTGCCCGTATAAACAGTTAATCATCAAGAAAACAAAATTAAT (SEQ ID NO. 265)42bpsGridiron-146AAAAGAAGATGATGAAACAAAGCCCCCTGCCTATTTCGGAAC (SEQ ID NO. 266)42bpsGridiron-147CTATTATTCTGAAACATGAAAAACGCTCAACAGTAGGCCTTA (SEQ ID NO. 267)42bpsGridiron-148ATTGAGAATCGCCATATTTAACACGGAATAAGTTTATTTTGT (SEQ ID NO. 268)42bpsGridiron-149CACAATCAATAGAAAATTCATATTCATTTCAATTACCTGAGC (SEQ ID NO. 269)42bpsGridiron-150CAGTACATAAATCAATATATGAATGCCACTACGAAGGCACCA (SEQ ID NO. 270)42bpsGridiron-151CTAAATCGTCGCTATTAATTAACCTGCTCCATGTTACTTAGC (SEQ ID NO. 271)42bpsGridiron-152AGCGCGAAACAAAGTACAACGATGGTTTGAAATACCGACCGT (SEQ ID NO. 272)42bpsGridiron-153TATAACTATATGTAAATGCTGCAAATATCGCGTTTTAATTCG (SEQ ID NO. 273)42bpsGridiron-154AAGAGGAAGCCCGAAAGACTTATGCAAATCCAATCGCAAGAC (SEQ ID NO. 274)42bpsGridiron-155TAGTGAATTTATCAAAATCATGGAAGCAAACTCCAACAGCTC (SEQ ID NO. 275)42bpsGridiron-156AGCTTCAAAGCGAACCAGACCAGGTCTGAGAGACTACCTTTT (SEQ ID NO. 276)42bpsGridiron-157AAAGAACGCGAGAAAACTTTTCTGACTATTATAGTCAGAAGC (SEQ ID NO. 277)42bpsGridiron-158CTCAAATGCTTTAAACAGTTCTAAGACGCTGAGAAGAGTCAA (SEQ ID NO. 278)42bpsGridiron-159AAACATAGCGATAGCTTAGATAGAAAACGAGAATGACCATAA (SEQ ID NO. 279)42bpsGridiron-160ATCAAAAATCAGGTCTTTACCTCAAATATATTTTAGTTAATT (SEQ ID NO. 280)42bpsGridiron-161TCATCTTCTGACCTAAATTTAGAGATTTGTATCATCCCCTGA (SEQ ID NO. 281)42bpsGridiron-162TAAATTGTGTCGAAATCCGCGATTTTCCCTTAGAATCCTTGA (SEQ ID NO. 282)The claims are not intended to be limited to the embodiments andexamples described herein.

1-9. (canceled)
 10. A composition comprising a plurality of immobileHolliday junction analogs linked together in a plurality of layeredframes, wherein each layer of frame has at least two DNA helices whichlie on opposite sides of the Holliday junction which also lie in thesame plane, and wherein said plurality of immobile Holliday junctionanalogs are linked together with a central strand of single-stranded DNAwithin said layer of frame.
 11. The composition of claim 10, whereineach layer of frame is independently selected from a hexagonal,rectangular, or parallelogram shape.
 12. The composition of claim 10,wherein said plurality of immobile Holliday junction analogs are linkedtogether in a frame having at least three layers.
 13. The composition ofclaim 10, wherein said plurality of immobile Holliday junction analogsare linked together in a frame having at least four layers.
 14. Thecomposition of claim 10, wherein said single stranded DNA comprisesM13mp18 DNA.
 15. The composition of claim 10, wherein the multi-layeredframe having at least three layers comprises a lattice defined by thelengths of said lattice, and the interstitial space confined by thelattice defines a cavity, wherein the lattice comprises at least threedouble-stranded DNA strands, each of which has an independent length.16. The composition of claim 13, wherein the length of thedouble-stranded DNA strands is from 21 nt to 63 nt.
 17. The compositionof claim 10, wherein the frame has at least four layers, comprises alattice defined by the lengths of said lattice, and the interstitialspace confined by the lattice defines a cavity, wherein the latticecomprises at least three separate lengths of double-stranded DNA. 18.The composition of claim 15, wherein the length of the double-strandedDNA strands is from 21 nt to 63 nt.
 19. A composition comprising aplurality of immobile Holliday junction analogs linked together to forma structure selected from: (a) an S-shaped structure as depicted in FIG.4A, (b) a spherical shaped structure as depicted in FIG. 4B, or (c) acorkscrew-shaped structure as depicted in FIG. 4C.